Circular dichroism study of supramolecular assemblies of guanosine 5'-monophosphate.

Concentration and temperature dependent studies of the circular dichroism of dianionic guanosine 5'-monophosphate (5'-GMP), where the cation was Na(+), K(+), or Rb(+) ion, were done to obtain information regarding the nature of the self-assembled 5'-GMP species in aqueous solution, including G-quartets and other structures. Concentrations in the 0.05-0.85 M range and temperatures in the 5-50 °C range were used. At the highest concentrations and 5 °C, Na(2)(5'-GMP) and K(2)(5'-GMP) formed a cholesteric phase, but Rb(2)(5'-GMP) did not. Evidence for antiparallel base stacking (stacked with opposite polarity; head to head) was observed for Rb(2)(5'-GMP) but not for the Na(+) or K(+) salts. This structure, believed to be G-quartets, had a melting temperature of 15 °C and dissociated into a second associated species as the temperature increased. The latter was present to the greatest extent at ∼40 °C, and it is characterized by a prominent negative CD band at 306 nm, which may be indicative of an X-DNA type of structure (an expanded G-quartet) or base-stacked monomers or dimers. The same negative band appeared at 310 nm in the CD spectra of K(2)(5'-GMP) and Na(2)(5'-GMP) but was much less intense in the latter case. K(2)(5'-GMP) also formed a noncholesteric phase containing at least two different species, one more stable at low temperatures and the other more stable at higher temperatures, similar to Rb(2)(5'-GMP). (1)H NMR spectroscopy was used to assist in the interpretation of the CD spectra.

Trapping of transcription factors with symmetrical DNA using thiol-disulfide exchange chemistry.

Green fluorescent protein (GFP) fused to the C-terminal 100 amino acids of CAAT enhancer binding protein (C/EBP) also containing an N-terminal (His)(6) tag (GFP-C/EBP) was used as a transcription factor model to test whether thiol-disulfide exchange reactions could be used to successfully purify transcription factors. A symmetrical dithiol oligonucleotide with dual CAAT elements was constructed with 5' and 3' thiols. Upon reduction, circular dichroism confirms it spontaneously anneals with its internally complementary sequence to form the hairpin structure: 5'-HS-GCAGATTGCGCAATCTGC 3'-HS-CGTCTAACGCGTTAGACG The specific GFP-C/EBP protein-DNA complex, formed in solution at nM concentrations, could then be recovered (trapped) via thiol-disulfide exchange with a disulfide thiopropyl-Sepharose and eluted with dithiothreitol. GFP-C/EBP was isolated from crude bacterial extract treated with iodoacetamide; DNA binding by GFP-C/EBP was unaltered by carboxyamidomethylation. Eluted GFP-C/EBP was of high purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein, after in-gel digestion with trypsin, was also characterized by capillary reversed-phase liquid chromatography-nano-electrospray ionization-tandem mass spectrometry and the results analyzed using MASCOT software searching of the non-redundant protein database. A score of 1874 with a sequence coverage of 51% encompassing both termini and internal sequences for the match with GFP-C/EBP confirms its identity and sequence. The method has high potential for the identification and characterization of transcription factors and other DNA-binding proteins.

Cyanide produced by human isolates of Pseudomonas aeruginosa contributes to lethality in Drosophila melanogaster.

Some Pseudomonas aeruginosa strains are cyanogenic, and cyanide may contribute to the bacterium's virulence. Using human isolates of P. aeruginosa, we have shown that Drosophila melanogaster suspended above cyanogenic strains become motionless and develop bradycardia and that flies injected with cyanogenic bacterial strains die more rapidly than those injected with noncyanogenic strains. Flies exposed to cyanogenic strains had high cyanide and low adenosine triphosphate (ATP) concentrations in body extracts, and treatment with a cyanide antidote equalized survival of flies injected with cyanogenic and noncyanogenic strains. P. aeruginosa PAO1 strain with a mutation in the hydrogen cyanide synthase gene cluster was much less toxic to flies than the parental cyanogenic strain or 2 knock-in strains. Transgenic flies overexpressing rhodanese, which detoxifies cyanide by converting it to thiocyanate, were resistant to cyanide and the increased virulence of cyanogenic strains. We conclude that D. melanogaster is a good model for studying cyanide produced by P. aeruginosa.

A fluorescence study of type I and type II receptors of bone morphogenetic proteins with bis-ANS (4, 4'-dianilino-1, 1'-bisnaphthyl-5, 5' disulfonic acid).

Crystallography studies on several members of the bone morphogenetic protein (BMP) receptors suggested that hydrophobic regions in these proteins play an important role in their structure and function. In the present study, the environment sensitive fluorescent probe 4, 4'-dianilino-1, 1'-bisnaphthyl-5, 5' disulfonic acid (bis-ANS) was used to study the hydrophobic regions of the extracellular domain of the type I and II receptors for bone morphogenetic proteins (ecBMPR-IB and ecBMPR-II). A single bis-ANS binding site per receptor molecule was found for both receptors, but the two receptors interacted with bis-ANS with distinctive characteristics. A significant shift in the emission maximum from 498 to 510 nm was detected when bis-ANS binds ecBMPR-IB, but a negligible change in the emission maximum was observed when the dye binds ecBMPR-II. Under identical reaction conditions, the maximum fluorescence intensities of the probe (I(max)) for the ecBMPR-IB and -II are 4.0 and 6.2 x 10(4) arbitrary units, respectively. The probe binds to ecBMPR-IB and -II with K(d)=11.0 and 17.5 microM, respectively. The bis-ANS modified site on both receptor types was not readily accessible to acrylamide quenching. Fluorescence energy transfer experiments further revealed close proximity between the tyrosine (in ecBMPR-IB) and the tryptophan residue (in ecBMPR-II) and the respective bis-ANS binding site in these receptors. The binding of bis-ANS did not alter the ligand binding activity of ecBMPR-IB, but enhanced that of ecBMPR-II. These results show that the bis-ANS-modified hydrophobic site on the ecBMPR-IB and -II molecules plays a different functional role.

Photolabeling of cardiolipin binding subunits within bovine heart cytochrome c oxidase.

Subunits located near the cardiolipin binding sites of bovine heart cytochrome c oxidase (CcO) were identified by photolabeling with arylazido-cardiolipin analogues and detecting labeled subunits by reversed-phase HPLC and HPLC-electrospray ionization mass spectrometry. Two arylazido-containing cardiolipin analogues were synthesized: (1) 2-SAND-gly-CL with a nitrophenylazido group attached to the polar headgroup of cardiolipin (CL) via a linker containing a cleavable disulfide; (2) 2',2''-bis-(AzC12)-CL with two of the four fatty acid tails of cardiolipin replaced by 12-(N-4-azido-2-nitrophenyl) aminododecanoic acid. Both arylazido-CL derivatives were used to map the cardiolipin binding sites within two types of detergent-solubilized CcO: (1) intact 13-subunit CL-containing CcO (three to four molecules of endogenous CL remain bound per CcO monomer); (2) 11-subunit CL-free CcO (subunits VIa and VIb are missing because they dissociate during CL removal). Upon the basis of these photolabeling studies, we conclude that (1) subunits VIIa, VIIc, and possibly VIII are located near the two high-affinity cardiolipin binding sites, which are present in either form of CcO, and (2) subunit VIa is located adjacent to the lower affinity cardiolipin binding site, which is only present in the 13-subunit form of CcO. These data are consistent with the recent CcO crystal structure in which one cardiolipin is located near subunit VIIa and a second is located near subunit VIa (PDB ID code referenced in Tomitake, T. et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 15304-15309). However, we propose that a third cardiolipin is bound between subunits VIIa and VIIc near the entrance to the D-channel. Cardiolipin bound at this location could potentially function as a proton antenna to facilitate proton entry into the D-channel. If true, it would explain the CcO requirement of bound cardiolipin for full electron transport activity.

Activation parameters for the spontaneous and pressure-induced phases of the dissociation of single-ring GroEL (SR1) chaperonin.

We investigated the dissociation of single-ring heptameric GroEL (SR1) by high hydrostatic pressure in the range 0.5-3.0 kbar. The kinetics were studied as a function of temperature in the range 15-35 degrees C. The dissociation processes at each pressure and temperature showed biphasic behavior. The slower rate (k1,obs) was confirmed to be the self-dissociation of SR1 at any specific temperature at atmospheric pressure. This dissociation was pressure independent and followed concentration-dependent first-order kinetics. The self-dissociation rates followed normal Eyring plots (In k1,obs/T vs. 1/T) from which the free energy of activation (deltaG++ = 22 +/- 0.3 kcal mol(-1)), enthalpy of activation (deltaH++ = 18 +/- 0.5 kcal mol(-1)), and entropy of activation (deltaS++ = -15 +/- 1 kcal mol(-1)) were evaluated. The effect of pressure on the dissociation rates resulted in nonlinear behavior (ln k2,obs vs. pressure) at all the temperatures studied indicating that the activation volumes were pressure dependent. Activation volumes at zero pressure (V++o) and compressibility factors (beta++) for the dissociation rates at the specific temperatures were calculated. This is the first systematic study where the self-dissociation of an oligomeric chaperonin as well as its activation parameters are reported.

Active rhodanese lacking nonessential sulfhydryl groups contains an unstable C-terminal domain and can be bound, inactivated, and reactivated by GroEL.

Mutation of all nonessential cysteine residues in rhodanese turns the enzyme into a form (C3S) that is fully active but less stable than wild type (WT). This less stable mutant allowed testing of two hypotheses; (a) the two domains of rhodanese are differentially stable, and (b) the chaperonin GroEL can bind better to less stable proteins. Reduced temperatures during expression and purification were required to limit inclusion bodies and obtain usable quantities of soluble C3S. C3S and WT have the same secondary structures by circular dichroism. C3S, in the absence of the substrate thiosulfate, is cleaved by trypsin to give a stable 21-kDa species. With thiosulfate, C3S is resistant to proteolysis. In contrast, wild type rhodanese is not proteolyzed significantly under any of the experimental conditions used here. Mass spectrometric analysis of bands from SDS gels of digested C3S indicated that the C-terminal domain of C3S was preferentially digested. Active C3S can exist in a state(s) recognized by GroEL, and it displays additional accessibility of tryptophans to acrylamide quenching. Unlike WT, the sulfur-loaded mutant form (C3S-ES) shows slow inactivation in the presence of GroEL. Both WT and C3S lacking transferred sulfur (WT-E and C3S-E) become inactivated. Inactivation is not due to irreversible covalent modification, since GroEL can reactivate both C3S-E and WT-E in the presence of GroES and ATP. C3S-E can be reactivated to 100%, the highest reactivation observed for any form of rhodanese. These results suggest that inactivation of C3S-E or WT-E is due to formation of an altered, labile conformation accessible from the native state. This conformation cannot as easily be achieved in the presence of the substrate, thiosulfate.

Dissociation of the single-ring chaperonin GroEL by high hydrostatic pressure.

We investigated the dissociation of single-ring heptameric GroEL (SR1) by high hydrostatic pressure in the range of 1-2.5 kbar. The kinetics of the dissociation of SR1 in the absence and presence of Mg2+, KCl, and nucleotides were monitored using light scattering. The major aim of this investigation was to understand the role of the double-ring structure of GroEL by comparing its dissociation with the dissociation of the single ring. At all the pressures that were studied, SR1 dissociates much faster than the GroEL 14mer. As observed with the GroEL 14mer, SR1 also showed biphasic kinetics and the dissociated monomers do not reassociate readily back to the oligomer. Unlike the GroEL 14mer, the observed rates for SR1 dissociation are independent of the concentrations of Mg2+ and KCl in the studied range. The effects of nucleotides on the observed rates, in the absence or presence of Mg2+ and KCl, are not very significant. The heterogeneity induced in the GroEL molecule with the double-ring structure by ligands such as Mg2+, KCl, and nucleotides is not observed in the case of SR1. This indicates that the inter-ring negative cooperativity in the double-ring GroEL has a major role in this regard. The results presented in this investigation demonstrate that the presence of a second ring in the GroEL 14mer is important for its stability in an environment where the functional ligands of the chaperonin are available.

Conformational heterogeneity is revealed in the dissociation of the oligomeric chaperonin GroEL by high hydrostatic pressure.

We investigated the dissociation of tetradecameric GroEL by high hydrostatic pressure in the range of 1-2.5 kbar. Kinetics of the dissociation of GroEL in the absence and presence of Mg(2+) and/or KCl were monitored using light scattering. All of the kinetics were biphasic in nature. At any given pressure, only monomers and 14mers were produced, and below 2.5 kbar, the 14mers only partially dissociated to monomers, which did not significantly reassemble on depressurization. Under identical reaction conditions, the observed dissociation rates decreased by only 2-fold when the concentration of GroEL was increased by 20-fold. At 2.5 kbar the observed rates decreased exponentially with the increase in [KCl] and reached a minimum at approximately 75mM. Similarly, the rates decreased with the increase in [Mg(2+)] and reached a minimum at approximately 3 mM Mg(2+). In the presence of saturating amounts of Mg(2+) (10 mM) and KCl (100 mM), the rates were much faster than with 10 mM Mg(2+) alone. The results could be rationalized in terms of the presence of GroEL heterogeneity, which could not be assessed easily by common techniques such as sedimentation velocity, HPLC, gel electrophoresis, and dissociation by chaotropes. This heterogeneity is evidence of subpopulations of GroEL that dissociate at different pressures. At low pressures, the oligomer without added Mg(2+) only partially dissociates to monomers, leading to an apparent plateau in the kinetics, whereas in the presence of Mg(2+) the species are converted to a tighter Mg(2+)-bound species, leading to a much slower dissociation process. The presence of KCl in the sample also leads to similar heterogeneity.